Dose-Related Structural and Functional Modifications of Mitochondria Are Induced In Vitro by Low Ozone Concentrations

In the last decades, ozone (O₃)-based medical treatments have become a widely applied complementary therapy for several pathological conditions. O₃ is administered at low dosages since the induction of a mild oxidative stress does not cause damage but stimulates the antioxidant cell response through the nuclear factor erythroid 2-related factor 2 (Nrf2). Mitochondria are sensitive to even mild oxidative stress, thus being a responsive target for O₃. This study aimed to evaluate the mitochondrial response to low O₃ doses used for medical treatments. As the skeletal muscle represents a primary target in local O₃ treatments, a murine non-tumoral muscle cell line was selected as an appropriate in vitro model. Transmission electron microscopy, biochemistry, and flow cytometry provided original information on the O₃ dose-dependent modifications of mitochondrial structural and molecular features. Low O₃ doses promoted an increase in mitochondrial area and in cristae extension, as well as an enhancement of the electron transport chain complexes and of antioxidant catalase and manganese-dependent superoxide dismutase. Nrf2 maintained its association with the outer mitochondrial membrane, thus exerting its protective role. All mitochondrial modifications were observed 24 h after treatment and disappeared after 48 h, demonstrating that cells promptly respond to the O₃-driven oxidative stress, effectively restoring homeostasis.